Immuno-Oncology Advances in Applied Cellular Analysis - 23

Advances in Applied Cellular Analysis * Continuous Live-Cell Analysis of the Immune-Tumor Axis

immunotherapy. Indeed, preclinical studies
indicate therapeutic benefit of anti-CD47 antibodies in both B-cell malignancies and solid
cancers.3,4 CCRF-CEM, a CD47-expressing lymphoblastic leukemia cell line, was prelabeled with a
non-perturbing pH-sensitive dye, pHrodo
(IncuCyte pHrodo Red Cell Labeling Kit for
Phagocytosis, 250ng.ml-1). Following 1h pretreatment with either anti-CD47 or IgG control,
CCRF-CEM (15K cells per well) were added directly
to pre-plated (4h) mouse bone marrow-derived
macrophages (BMDMs, 10K). IncuCyte images
(20x) were taken every 15 min and analyzed for
the appearance of red fluorescence objects over
time (internalized CCRF-CEM cells in the acidic
phagosome of the BMDM). Anti-CD47 caused
marked and rapid (<1h), concentration-dependent cellular phagocytosis with a threshold concentration of 40ng.ml-1 (Figure 6). BMDMs could
be clearly seen to engulf CCRF-CEMs to trigger
the appearance of the red signal. IgG had little or
no effect and if BMDMs were omitted from the
assay no signal was observed. Similar observations were made using anti-CD47 Ab in assays
with J774.1 macrophages and pHrodo labeled
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Figure 6. IncuCyte continuous live-cell assay for anti-CD47 Ab-mediated cellular engulfment of CCRF-CEM
by human bone marrow-derived macrophages.
Timecourse (A) and Area Under Curve (B) analysis. Fluorescent Area = total red object area (x103 (μm²/ Image)
and Area Under Time Curve units are x103 (0-4h). Values are mean ± SD (n=4).

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Immuno-Oncology Advances in Applied Cellular Analysis

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